This invention relates to the enhancement of whole cell biosynthesis of precursors of chorismate and compounds biosynthetically derived therefrom. More particularly, this invention relates to overproduction in whole cells of enzymes that catalyze the biosynthetic production of aromatic compounds.
Chorismate is an intermediate in biosynthetic pathways that lead to the production of aromatic compounds such as phenylalanine, tryptophan, tyrosine, folate, melanin, ubiquinone, menaquinone, prephenic acid (used in the production of the antibiotic bacilysin) and enterochelin. Because of the large number of biosynthetic pathways that depend from chorismate, the biosynthetic pathway utilized by organisms to produce chorismate is often known as the "common aromatic pathway". The common aromatic pathway (sometimes referred to as the "shikimate pathway" because shikimate was the first identified intermediate in the pathway) is generally considered to begin with the DAHP-synthase catalyzed condensation of the precursor compounds erythrose 4-phosphate and phosphoenolpyruvate to form 3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) and . to end with the production of chorismate.
Efficient and cost-effective biosynthetic production of chorismate and its biosynthetic derivatives require that carbon sources such as glucose, lactose, galactose, or other sugars be converted to the desired product with high percentage yields. Accordingly, it is valuable from the standpoint of industrial biosynthetic production of aromatic compounds or other biosynthetic derivatives of chorismate to increase the influx of carbon sources into and through the common aromatic pathway thereby enhancing whole cell biosynthesis of chorismate and its derivatives.
The present invention provides for the enhanced commitment of cellular carbon sources to enter and flow through the common aromatic pathway by transferring into host cells genetic elements comprising a tkt gene and optionally other genetic elements encoding enzymes that direct carbon flow into or through the common aromatic pathway. The genetic elements can be in the form of extrachromosomal plasmids, cosmids, phages, or other replicons capable of carrying at least the gene tkt for expression in a host cell. The tkt gene codes for the enzyme transketolase, which catalyzes the conversion of carbon source D-fructose 6-phosphate to D-erythrose 4-phosphate, a necessary precursor compound for the common aromatic pathway. Overproduction of transketolase in tkt transformed cells has been found to provide an increased flow of carbon resources into the common aromatic pathway relative to carbon resource utilization in whole cells that do not harbor such genetic elements.
In addition to transketolase, other enzymes that catalyze steps of the common aromatic pathway or that catalyze production of compounds input into the common aromatic pathway can be overexpressed in whole cells to increase the efficiency of carbon conversion and biosynthetic throughput of the common aromatic pathway. The present invention also provides for transfer of genetic elements comprising the tkt gene, the gene coding for DAHP synthase (aroF in E. coli), an enzyme required for condensation of phophoenolpyruvate and erythrose 4-phosphate, the gene coding for 3-dehydroquinate synthase (aroB in E. coli), or other genes encoding enzymes that catalyze reactions in the common aromatic pathway. In a preferred aspect of the invention, an enzyme that produces a precursor to compounds input into the common aromatic pathway (such as transketolase) as well as one or more enzymes that catalyze biosynthetic steps of the common aromatic pathway are encoded on one or more plasmids transferred into a host cell. As a result of this transfer of genetic element(s), more carbon enters and moves through the common aromatic pathway relative to wild type whole cells not containing the genetic elements of the present invention.
Generally, the present invention enhances expression in a host cell of transketolase and other enzymes catalyzing conversion of substrates in the common aromatic pathway relative to wild type host cell expression of the enzymes. This enhanced expression of genes encoding the enzymes is attained either by the transfer and stable incorporation of extrachromosomal genetic elements into the host cell or by the transfer of the genetic elements into the genome of the host cell. The expressed gene products are enzymes configured to provide appropriate catalytic sites for substrate conversion of common aromatic pathway compounds.